Abstract

Sepsis is characterized by systemic infection that causes multiple organ dysfunction and is associated with a high mortality rate in the paediatric intensive care unit. In the present study, we investigated the effect of geraniol on sepsis in neonatal rats. Sepsis was induced in neonatal rats by intraperitoneal (i.p.) inoculation with almost 6,000 colony forming units of Escherichia coli. The rats were treated with geraniol (25 or 50 mg/kg i.p.) 1 h after the administration of E. coli. Survival rate over 30 h, bacterial load, blood parameters, and inflammatory cytokines were measured. Histopathological analysis and qRT-PCR were performed on rat liver tissues. Geraniol improved the survival rate and sepsis severity in neonatal sepsis rats. Inflammatory cytokine levels, liver function test results, and blood parameters were improved in the geraniol-treated compared to sepsis group. Geraniol attenuated mRNA expression of calprotectin (S100A8/S100A9) and TLR-4 in the liver tissue of neonatal sepsis rats. Therefore, geraniol reduced bacterial load in body fluids and inflammatory mediators in neonatal sepsis rats by inhibiting calprotectin and TLR-4 pathways.

1. Introduction

Sepsis refers to a systemic inflammation or infection, and is a major cause of mortality in intensive care units worldwide ¹. The mortality rate of sepsis is higher in the neonatal age group compared to other age groups due to the weak immune response of the former ². Bacterial infections in adults are usually controlled by the immune system; however, the weaker immune system of neonates fails to control the infection, which may result in a severe systemic inflammatory response ³. Despite advances in treatment, sepsis remains the leading cause of death among patients admitted to neonatal intensive care units. The management of sepsis includes supportive care and antibiotics. Inflammatory cytokines, such as interleukin (IL)-6 and tumour necrosis factor (TNF)-α, are increased in severe sepsis and contribute to the development of complications, i.e., multiple organ dysfunction ⁴. Therefore, anti-inflammatory drugs are also used to treat neonatal sepsis.

The immune system is involved in the pathogenesis of sepsis, and activation of immune cells increases cytokine release ⁵. Immune cells interact with pathogens to release calprotectin, which is stored in neutrophil cytoplasm ⁶. Calprotectin is a biomarker of sepsis and reduced calprotectin levels are associated with attenuation of sepsis ⁷.

Geraniol is an acyclic monoterpenoid, which is an essential oil molecule isolated from Cymbopogon winterianus, Dracocephalum moldavica, Monarda fistulosa, and Rosa damascene ⁸. Geraniol has anti-diabetic, anti-allergic, hepatoprotective, anti-inflammatory, anti-diarrheal, and antioxidant activities ^{9, 10, 11, 12, 13}. Geraniol regulates the immune system to produce immunomodulatory, anti-Parkinson’s, and anti-cancer effects ^{14, 15}. In the present study, we evaluated the protective effect of geraniol against sepsis in neonatal rats.

2. Material and Methods

Mother and new-born pups were housed under controlled conditions in a pathogen-free environment. The animal experiments were approved by the Animal Ethical Committee of Southern Campus of Guang’anmen Hospital, China (IAEC/SC-GH/02/2020).

Sepsis was induced in neonatal rats by intraperitoneal (i.p.) inoculation with almost 6,000 colony forming units (CFU) of Escherichia coli. The neonatal rats were randomized into four groups: control (without sepsis), sepsis (vehicle treatment), geraniol 25 (treated with 25 mg/kg i.p. 1 h after sepsis induction), and geraniol 50 (treated with 50 mg/kg i.p. 1 h after sepsis induction) groups. The bacterial level was determined by quantitative blood cultures before and after 5 h of treatment. Clinical signs of sepsis were also recorded, including decreased food intake, huddling, reduced spontaneous activity, increased body temperature, and lethargy.

Neonatal rats were observed for 30 h to determine the survival rate in E. coli- and geraniol-treated rats. Appearance, Level of consciousness, Activity, Response to stimulus and eyes were recorded. Clinical signs were rated on 5-point scales (range: 0–4) with a maximum total score of 20.

Sterile phosphate-buffered saline was injected into the peritoneum and aspirated to collect peritoneal fluid. Peritoneal fluid and blood were serially diluted. Lung and liver were isolated from the rats, homogenized, and centrifuged. The supernatant was collected and serially diluted in saline. Lung and liver tissues, peritoneal fluid, and blood were incubated on an agar plate to determine the bacterial load. Bacterial colonies were estimated as log CFU/g of tissue for organs and log CFU/mL of blood.

Plasma aspartate transaminase (AST) and alanine transaminase (ALT) levels (U/L) in septic rats were determined using a semi-automated analyzer. Organ tissues were homogenized and centrifuged at 4^oC for 5 min at 5,000 × g. The levels of inflammatory cytokines, including IL-1β, IL-6, interferon-γ, and TNF-α, in the supernatant were estimated using ELISA (Thermo Fisher Scientific, Waltham, MA, USA).

The Sysmex XT-2000i haematology analyser (Sysmex Corp., Kobe, Japan) was used to analyse blood parameters, including differential count, white blood cell (WBC) count, and haemoglobin level. Immunofluorescence staining was performed to estimate the CD4+/CD8+ T lymphocyte ratio using flow cytometry.

Liver and lung tissues were fixed in 10% formalin solution and embedded in molten paraffin to prepare wax cubes. Tissues were cut into 3-μm-thick slices using a microtome and stained with hematoxylin and eosin. Histopathological changes were observed using a CX41 microscope (Olympus, Tokyo, Japan).

RNAiso Plus reagent (ThermoFisher Scientific Ltd, USA) was used to extract total RNA according to the manufacturer’s instructions. Reverse transcriptase was used to synthesize cDNA from total RNA (1 μg). The SYBR-RT PCR kit was used to perform quantitative real-time PCR (qRT-PCR). The expression levels of calprotectin (S100A8/S100A9), TLR-4, and IL-6 mRNA were estimated in tissue homogenates using qRT-PCR.

The data are expressed as mean ± standard error of the mean (SEM; n = 8). Data were analysed by one-way analysis of variance, followed by post hoc Dunnett’s test, using GraphPad Prism software (version 6.1; GraphPad Software Inc., San Diego, CA, USA). P-values < 0.05 were considered statistically significant.

3. Results

Geraniol use significantly affected the clinical score and survival percentage of septic neonatal rats (Figure 1A & B). The percentage survival of rats was reduced by up to 10% in the sepsis compared to control group (i.e., 100%). Moreover, the percentage survival of geraniol-treated septic rats improved by up to 70% (Figure 1A). The clinical score was significantly increased (p < 0.01) by up to 20-fold in the septic compared to control group. The clinical score was significantly reduced in the geraniol-treated group compared to the sepsis group (p < 0.01) (Figure 1B).

The bacterial load was determined in the body tissues and fluids of geraniol-treated neonatal rats (Figure 2). The bacterial load was significantly increased in the body fluids (peritoneal fluid and blood) and tissues (liver and lung) of the septic compared to the control group (p < 0.01). However, geraniol treatment significantly reduced the bacterial load in the body fluids (peritoneal fluid and blood) and tissues (liver and lung) of septic neonatal rats.

Figure 3 summarizes the effects of geraniol on the levels of inflammatory mediators in tissue homogenates of septic neonatal rats. The levels of inflammatory cytokines, including INF-γ, IL-1β, IL-6, and TNF-α, significantly increased in the tissue homogenates of the sepsis compared to control group. However, treatment with geraniol reduced the cytokine levels in tissue homogenates of septic neonatal rats.

Figure 4 depicts the effects of geraniol on liver function, including plasma liver protein, in neonatal rats. Plasma AST and ALT levels were significantly increased in the plasma of the sepsis compared to control group (p < 0.001). Plasma AST and ALT levels were significantly reduced in the geraniol-treated group in a dose-dependent manner (p < 0.01; p < 0.001).

The red blood cell (RBC) count, WBC count, haemoglobin, haematocrit, neutrophil and lymphocyte counts, and CD4+/CD8+ ratio were measured in the geraniol-treated septic neonatal rats. The RBC count, haematocrit, haemoglobin, and CD4+/CD8+ ratio were significantly reduced in the sepsis compared to control group (p < 0.01); these changes were attenuated by geraniol treatment in sepsis rats.

Figure 6A & B shows the histopathological changes in lung and liver tissues of geraniol-treated septic neonatal rats revealed by hematoxylin and eosin staining. The lung and liver tissues of the control group appeared normal on histopathology. Lung tissues from the sepsis group showed histopathological changes, including pneumonitis characterized by diffuse infiltration of inflammatory cells in alveoli. Moreover, liver hepatocytes showed prominent hydropic degenerative changes in the sepsis group. However, geraniol treatment attenuated these pathological changes in the lung and liver tissues of septic neonatal rats.

We performed qRT-PCR to determine the mRNA expression levels of S100A8, S100A9, TLR-4, and IL-6 in the liver tissues of geraniol-treated septic neonatal rats (Figure 7). The mRNA expression levels of S100A8, S100A9, TLR-4, and IL-6 were significantly increased in the liver tissue of the sepsis compared to control group (p < 0.01). The mRNA expression levels of S100A8, S100A9, TLR-4, and IL-6 were significantly reduced in the liver tissue of the geraniol-treated sepsis group.

4. Discussion

Sepsis is a manifestation of systemic bacterial infection characterized by immune activation. Phagocytes are activated in response to the recognition of infection by pattern recognition receptors ¹⁶. Moreover, damage-associated molecular patterns also provide endogenous signals to activate phagocytes ¹⁷. Inflammatory stimuli and stress stimulate the release of S100A8 and S100A9 proteins by phagocytes ¹⁸. Adhesion molecules and pro-inflammatory cytokines are induced by S100A8 and S100A9 proteins, which have pro-inflammatory properties ¹⁹. The levels of S100A8 and S100A9 proteins are elevated in sepsis; regulating the S100A8 and S100A9 proteins could be a potential target for the treatment of inflammation and sepsis. Therefore, we studied the effects of geraniol on the regulation of S100A8 and S100A9 proteins in sepsis.

Sepsis is a multifactorial disorder with several treatment targets, such as TLR-4 ²⁰. In septic shock, TLR-4 activation increases cytokine release, which exacerbates systemic inflammation and may even lead to death ²¹. Liver Kupffer cells are also activated during sepsis, which causes cytokine release ²². The significant increase in cytokines causes cellular injury, leading to multiple organ dysfunction. Our results showed that cytokine and TLR-4 expression were increased in the sepsis compared to control group, and these effects were attenuated by geraniol treatment.

Blood parameters, such as RBC, WBC, haemoglobin, haematocrit, neutrophil and lymphocyte counts, and the CD4+/CD8+ ratio, were altered in septic rats, consistent with previous studies ²³. Anaemia is a severe complication of sepsis characterized by reduced haemoglobin and haematocrit levels. Moreover, the antigenic properties of microorganisms contribute to an increase in the WBC count and decrease in the CD4+/CD8+ ratio in sepsis ²⁴. Sepsis is managed by correcting these blood parameters. In the current study, geraniol treatment improved the altered blood parameters.

In sepsis, histopathological changes occur in liver tissue due to injury and inflammation. Liver dysfunction in sepsis is evidenced by increased ALT and AST levels. The ALT and AST levels were significantly reduced in our geraniol-treated compared to sepsis group.

5. Conclusion

Geraniol reduced inflammatory mediators and bacterial load in body fluids in septic neonatal rats by inhibiting calprotectin and the TLR-4 pathway. Geraniol also improved blood parameters and liver function in septic neonatal rats.

Acknowledgements

The authors are thankful to the Southern Campus of Guang’anmen Hospital, China, for providing the facilities necessary to conduct the study.

References